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Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEP-CaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8 + T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The rabies virus envelope glycoprotein (RVGP) is the main antigen of rabies virus and is the only viral component present in all new rabies vaccines being proposed. Many approaches have been taken since DNA recombinant technology became available to express an immunogenic recombinant rabies virus glycoprotein (rRVGP). These attempts are reviewed here, and the relevant results are discussed with respect to the general characteristics of the rRVGP, the expression system used, the expression levels achieved, the similarity of the rRVGP to the native glycoprotein, and the immunogenicity of the vaccine preparation. The most recent studies of rabies vaccine development have concentrated on in vivo expression of rRVGP by viral vector transduction, serving as the biotechnological basis for a new generation of rabies vaccines.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Vaccination is the sensitization process of the immune system against any pathogen. Generally, recombinant subunit vaccines are considered safer than attenuated vaccines. As whole pathogenic organisms are used in the immunization process, the attenuated vaccines are considered more risky than subunit vaccines. Rabies is the oldest known zoonosis which spreads through a neurotropic Lyssavirus primarily mediated through infected canine bites. Rabies causes worldwide loss of more than 60,000 human lives every year. Animal vaccination is equally important to check the transmission of rabies into humans. Rabies oral vaccination can be a good alternative where multiple booster and priming regimens are required while the painful vaccination process can continue for long durations. Introduction of oral vaccines was made to ease the discomfort associated with the mode of introduction of conventional vaccines into the body. Although the rabies oral vaccine can substantially reduce the cost of vaccination in the developing countries, mass immunization programs need larger quantities of vaccines which should be delivered at nominal cost. Expression of recombinant antigen proteins in E. coli is often not viable because of lack of post-translational modifications and folding requirements. Though yeast and insect cell line expression systems have post-translational processing and modifications, significantly different immunological response against their post-translational modification pattern limits their deployment as an expression system. As an alternative, plants are emerging as a promising system to express and deliver wide range of functionally active biopharmaceutical product at lower cost for mass immunization programs. As generation of vaccine antigenic proteins in plant systems are cheaper, the strategy will benefit developing countries where this disease causes thousands of deaths every year. In this chapter, we will discuss about our efforts toward development of oral rabies vaccine and the methodological steps involved during this procedure in detail.

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.

In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Vero cells are utilized for production of rabies vaccine. This study deals with the optimize quantity media require for the rabies vaccine production in the smooth roller surface. The rabies virus (Pasteur vaccine strain) is infected to monolayer of the various experimented bottles. To analyze the optimal quantity of media for the production of rabies viral harvest during the process of Vero cell derived rabies vaccine. The trials are started from 200 to 400 mL (PTARV-1, PTARV-2, PTARV-3, PTARV-4 and PTARV-5). The samples are taken in an appropriate time intervals for analysis of In Process Quality Control (IPQC) tests. The collected viral harvests are further processed to rabies vaccine in a pilot level and in addition to scale up an industrial level. Based on the evaluation the PTARV-2 (250 mL) show highly encouraging results for the Vero cell derived rabies vaccine production.

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.

The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6 mM to the viral harvest at 37 degrees C, followed by a second dose of BEI at 24 h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4 mM) was able to inactivate the rabies virus within 30 h and the routine concentration (1.6 mM) of BEI is effective in inactivating rabies virus within 18 h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed.

A new rabies vaccine was developed from Vero cells adhered to microcarriers, cultivated in a bioreactor in serum-free medium and infected with the PV/VERO-Paris rabies virus strain. The viral suspensions were concentrated by tangential filtration, purified by chromatography and inactivated with beta-propiolactone. In immunogenicity studies performed in mice immunized with three doses of the new vaccine (seven batches) and the commercial Verorab and HDCV, mean titers of neutralizing antibodies of 10.3-34.6, 6.54 and 9.36 IU/ml were found, respectively. The vaccine presented stability during 14 months at 2-8 degrees C, 30 days at 37 degrees C and 8 h at 45 degrees C. The use of serum-free medium facilitated the downstream process leading to residual cellular DNA values <22.8 pg per dose of vaccine in all produced batches. The effective immunogenicity induced in mice by this vaccine, the degree of purity of the product, the high antigen yield and the reduction of the cost of the product due to the virus production and purification processes, makes this technology very important for countries where rabies presents a great public health problem.

Objective: To construct and characterize recombinant adenoviruses containing glycoprotein (GP) gene from rabies virus CVS-N2C strain., Methods: To obtain the recombinant adenovirus by pAdEasy system, identify recombinant virus with cDNA sequencing, Northern blot, Dot blot, Western blot and challenge-protection experiment of mice., Results: Recombinant adenovirus showed typical adenovirus morphological characteristics; the viral genome was stable; GP specific mRNA and presence of glycoprotein were determined in rAdGPcvs and rAdGPcvs' infected cells. The glycoprotein produced by recombinant adenovirus had a molecular mass of 66,000, which was similar to that of natural glycoprotein. In the group of rAdGPcvs immunized mice, 87.5%-100% of mice survived from a 35.8LD50/38.0LD50 lethal rabies intracerebral challenge. Finally 73.3%-83.3% of the mice that had received eAdGPcvs survived, and all the Ad5 immunized mice succumbed to rabies., Conclusion: Recombinant adenovirus rAdGPcvs and rAdGPcvs' hold great potential to be developed as recombinant rabies vaccines, and at the same time, it is actually the first study that on high neuropathogenicity rabies CVS-N2C glycoprotein based adenoviral recombinants.

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations., (Copyright 2002 Elsevier Science Ltd.)

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.

Objective: To improve the safety of recombinant vaccinia rabies virus, the non-replicating recombinant vaccinia rabies virus VTKRG delta CK was constructed., Methods: The rabies glycoprotein(RG) gene was introduced into the TK locus of Chinese Tiantan strain of vaccinia virus (VTKRG), and a fragment between fragment C and fragment K involving vaccinia virus virulence and host-range related genes were deleted by homologous recombinant., Results: The ability to replicate in primary chick chick embryo fibroblast(CEF) and dramatically reduced replicating capability in mammalian cells of the virus were observed, whereas VTKRG delta CK could express high level of RG efficiently. The expression of RG could be detected in CEF by indirect immunofluorescence assay and Western blot analysis. The VTKRG delta CK could elicit neutralizing antibody against CVS rabies virus and protect the animals from lethal rabies virus challenge in inoculated mice., Conclusion: VTKRG delta CK possesses immunogenicity and higher safety.

By producing the rabies vaccine from cell culture, this vaccination has become safe, with minimal postvaccinal reactions. The first vaccine according to this technology was produced by Pavle (Paul) Fenje, former chief of department of the Pasteur Institute in Novi Sad. Many cell cultures have been introduced so far for the rabies virus multiplication: primary hamster kidney, fetal bovine kidney, chick embryo, continuous cell line monkey kidney (VERO), human diploid cell (HDC), etc. Some possibilities of an economical rabies vaccine production from a continuous BHK-21 cell line have been discussed and recommended.

Nerve tissue derived Semple and suckling mouse brain rabies vaccines are still widely used. Patients who experience a new rabies exposure and who were given such vaccines decades earlier are not uncommon in rabies endemic countries. The World Health Organization recommends that persons who have had a previous course of a potent tissue or avian culture rabies vaccine and are reexposed, be given booster infections on days 0 and 3 without rabies immune globulin. Persons who have had previous pre- or postexposure vaccination with a vaccine of unproven potency, should receive a full course of tissue or avian cell vaccine and immune globulin in the event of a new exposure. This study evaluated the immune response in 98 Thai patients who gave a history of rabies postexposure treatment with Semple or suckling mouse brain vaccines 10-50 years previously. The majority (81) had an anamnestic response and developed neutralizing antibodies that were above the recommended minimal acceptable level (0.5 IU ml-1) on day 7. This suggests that they still had immunological memory. A minority of 18% had antibody titers below this level on day 7. However, they all developed titers above 0.5 IU ml-1 on days 14 and 30. Failure to have an accelerated response to revaccination by day 7 did not appear to be related to age or time elapsed since previous nerve tissue derived vaccine administration. It was not possible to predict which subject will or will not have an acceptable level of antibody before day 14. Rabies exposed patients who give a prior history of vaccination with an unknown or nerve tissue derived vaccine should therefore be treated as if they had never been vaccinated.

The prevalence of wildlife rabies throughout the world and the continued spread of this disease in North America highlights the need for oral vaccines which may be used safely and effectively to vaccinate a number of species that are reservoirs or vectors of rabies. We have previously shown that AdRG1, a replication competent recombinant human adenovirus type 5 (Ad5) expressing a rabies glycoprotein (RG), can induce immunity to rabies in rodent, canine, and skunk model systems. To improve the Ad5 vector system as a potential oral vaccine, we have constructed additional Ad5 recombinant vectors and compared RG expression in cell culture and immunogenicity in animals. Two new replication competent vectors are compared. AdRG1.3, which carries RG with accompanying SV40 poly A addition sequences within an E3 deletion, and AdRG4, which has RG in the E3 deletion but under the control of an exogenous Ad2 major late promoter, both express higher levels of RG in permissive cell culture than did AdRG1 and both elicit high levels of serum anti-rabies antibodies by parenteral or oral routes in animals. AdRG1.3 may be a more effective vaccine vector in species which are non-permissive for the replication of human Ad5.

For the control of urban rabies in developing countries, the SAD-virusstrain was used in the Göttingen two-step Bioreactorsystem as a model-virus for oral vaccine production. The quality of the antigen had been tested by challenge. In the Bioreactorsystem, controlled by computer, a high standard of cell quality and cell density up to 6 x 10(6) cells/ml had been maintained. The high cell quality is correlated to high proliferation of rabies virus. Virus titres up to 10(8.5) pfu/ml were reached. The protectivity of the antigen had been tested by challenge. In comparison to a commercial vaccine with a PD50 of 10(-1.92) the SAD-Antigen of the Göttingen Bioreactorsystem reached a PD50 of 10(-1.63) by a challenge doses of 70-75 MLD50.

BHK-21 cells were grown in microcarriers in the CELLIGEN CL 50 bioreactor to produce a stock of rabies veterinary virus vaccine PV (Pasteur virus) strain. Perfusion mode operation of this bioreactor produced between two- and fourfold larger yields (cells/ml) than traditional stationary cell culture systems (i.e., Blake, and Roller bottles or cell factory multitrays). The method employed harvested 281 of rabies virus in 200 h (infectivity titer 0.6 +/- 1.4 x 10(7) LD50 per ml) in a single operation. The risk of contamination is thus reduced when compared with traditional stationary methods which, in order to obtain the same amount of virus, would require the operation of 285 Blake bottles, or 143 Roller bottles, or 15 Cell Factory multitrays (10 trays). By perfusion mode operation of the bioreactor, 89% of the cell culture medium was recovered as vaccinal virus, which contrasts with the yield of only 50-59% using traditional cell culture systems. On the other hand, only 925 ml of fetal serum was required to obtain the 281 of rabies virus harvest as compared to the 3420 ml required by traditional methods.

Although the RC-HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.

The live modified rabies virus vaccine strain SAG-2 was selected from SADBerne in a two step process employing anti-rabies glycoprotein monoclonal antibodies. The first two nucleotides coding for the amino acid in position 333 of the rabies glycoprotein are mutated. Arginine at position 333, which is associated with rabies pathogenicity, was substituted first by lysine and then by glutamic acid. The two nucleotide differences at position 333 in SAG-2 to any of six possible arginine triplets translated into excellent genetic stability and apathogenicity for adult mice, foxes, cats and dogs. The vaccination of foxes and dogs by the oral route provided protection against a lethal challenge with rabies virus.

The rabies virus surface glycoprotein was synthesized in Saccharomyces cerevisiae using an expression vector which contains an inducible promoter from the copper metallothionein gene. The rabies G protein was also expressed constitutively in yeast when cloned under control of the triose dehydrogenase promoter. Polypeptides of 65-68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by yeast transformants as detected by immunoblotting with rabies specific antiserum. In addition, these polypeptides were immunoprecipitated with several rabies G-specific monoclonal antibodies which neutralize virus infectivity. The recombinant rabies G proteins were glycosylated and associated with membranes in yeast. When injected into guinea pigs, yeast extracts containing the rabies G protein protected animals from lethal rabies virus challenge when administered intramuscularly. However, the same material did not protect mice from a lethal rabies intracerebral challenge.

Vaccination is one of the major preventive measures against infectious diseases. With the exception of the hepatitis B vaccine, the vaccines in use today are produced from the infectious agents themselves, either by attenuation or inactivation. Although these products have been successful in controlling many diseases, there are several reasons why efforts are being made to improve their quality. In addition there are some infectious diseases for which vaccines are not available because the causal agents cannot be grown in sufficient quantities. New approaches will be required to obtain effective vaccines against these diseases. In this paper, these approaches to the design of new vaccines are described using hepatitis B, rabies and foot-and-mouth disease as examples.